Humanized dipeptidyl-peptidase iv (dpp4) animals

ABSTRACT

Non-human animals comprising a human or humanized DPP4 nucleic acid sequence are provided. Non-human animals that comprise a replacement of the endogenous Dpp4 gene with a human or humanized DPP4 gene, or non-human animals comprising a human or humanized DPP4 gene in addition to the endogenous Dpp4 gene are described. Non-human animals comprising a human or humanized DPP4 gene under control of human or non-human DPP4 regulatory elements is also provided, including non-human animals that have a replacement of non-human Dpp4-encoding sequence with human DPP4-encoding sequence at an endogenous non-human Dpp4 locus. Non-human animals comprising human or humanized DPP4 gene sequences, wherein the non-human animals are rodents, e.g., mice or rats, are provided. Methods for making and using the non-human animals are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/295,955, filed Oct. 17, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/723,855, filed May 28, 2015, now U.S. Pat. No.9,497,945, which claims priority to U.S. Provisional Patent ApplicationNo. 62/005,476, filed May 30, 2014, U.S. Provisional Patent ApplicationNo. 62/051,626, filed Sep. 17, 2014, and U.S. Provisional PatentApplication No. 62/072,692, filed Oct. 30, 2014, the disclosures of eachof which are incorporated by reference herein in their entireties.

INCORPORATION BY REFERENCE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CFR) of the Sequence Listing (file name:47206_501C02US_Sequence_Listing_122016, date created: Dec. 30, 2016,size: 31, 792 bytes).

FIELD OF INVENTION

Non-human animals comprising nucleic acid sequences encoding adipeptidyl peptidase IV (DPP4) protein that comprise a human sequence.Transgenic non-human animals comprising a DPP4 gene that is human inwhole or in part. Non-human animals that express human or humanized DPP4proteins. Methods for making and using non-human animals comprisinghuman or humanized DPP4 nucleic acid sequences.

BACKGROUND

Dipeptidyl peptidase IV (DPP4) is a therapeutic target for the treatmentof a variety of human diseases, disorders and conditions, including, forexample, hyperglycemia (see, e.g., Gerich (2013) Pathogenesis andManagement of Postpandrial Hyperglycemia: Role of Incretin-BasedTherapies, Intl. J. Gen. Med. 6:877-895) and Middle East respiratorysyndrome coronavirus (MERS-CoV) infection (see, e.g., Raj et al. (2013)Dipeptidyl Peptidase 4 is a Functional Receptor for the Emerging HumanCoronovirus-EMC, Nature 495(7440):251-254).

The evaluation of the pharmacokinetics (PK) and pharmacodynamics (PD) oftherapeutic molecules that specifically target human DPP4 protein areroutinely performed in non-human animals, e.g., rodents, e.g., mice orrats. However, the PD of such molecules cannot properly be determined incertain non-human animals if these therapeutic molecules also do nottarget the endogenous Dpp4 protein.

Moreover, the evaluation of the in vivo therapeutic efficacy of humanDPP4-specific small molecule, peptide or protein (i.e., biologic)antagonists in non-human animal models of diseases is problematic incertain non-human animals in which the species-specific antagonist doesnot interact with the endogenous Dpp4 protein. Furthermore, theevaluation of the in vivo therapeutic efficacy of small molecule,peptide or protein (i.e., biologic) antagonists that target moleculesthat specifically interact with human DPP4 protein is also problematicin certain non-human animals in which the therapeutic target moleculeitself does not interact with the endogenous Dpp4 protein.

Accordingly, there is a need for non-human animals, e.g., rodents, e.g.,mice or rats that comprise a human or humanized DPP4 gene. For example,there is a need for non-human animals, e.g., rodents, e.g., mice orrats, in which the Dpp4 gene of the non-human animal is humanized inwhole or in part or replaced (e.g., at the endogenous non-human loci)with a human DPP4 gene comprising sequences encoding human or humanizedDPP4 protein.

There is also a need for non-human animals comprising a DPP4 gene (e.g.,human or humanized) in which the DPP4 gene is under control of non-humanregulatory elements (e.g., endogenous regulatory elements), for example,in the 5′ flanking region, e.g., promoter and enhancer(s), or in the 3′untranslated region, of the DPP4 gene.

There is also a need for non-human animals comprising a DPP4 gene (e.g.,human or humanized) in which the DPP4 gene is under control of humanregulatory elements, for example, in the 5′ flanking region, e.g.,promoter or enhancer(s), or in the 3′ untranslated region, of the humanDPP4 gene.

There is also a need for humanized non-human animals that express humanor humanized DPP4 protein on the surface of immune cells, e.g., T cells,and/or on the surface of cells in one or more tissues, e.g., placenta,kidney, lung, liver, skeletal muscle, heart, brain and/or pancreas, at alevel similar to that of Dpp4 protein on the surface of immune cells,e.g., T cells, and/or on the surface of cells in one or more tissues,e.g., placenta, kidney, lung, liver, skeletal muscle, heart, brainand/or pancreas, of an age-matched non-human animal that expressesfunctional Dpp4 protein, but does not comprise the human or humanizedDPP4 gene.

In addition, there is a need for humanized non-human animals thatexpress human or humanized DPP4 protein on the surface of immune cells,e.g., T cells, and/or on the surface of cells in one or more tissues,e.g., placenta, kidney, lung, liver, skeletal muscle, heart, brainand/or pancreas, at a level higher than or lower than that of Dpp4protein on the surface of immune cells, e.g., T cells, and/or on thesurface of cells in one or more tissues, e.g., placenta, kidney, lung,liver, skeletal muscle, heart, brain and/or pancreas, of an age-matchednon-human animal that expresses functional Dpp4 protein, but does notcomprise the human or humanized DPP4 gene.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles, electronic databaseentries, etc.) are referenced. The disclosure of all patents, patentapplications, and other publications cited herein are herebyincorporated by reference in their entirety for all purposes.

SUMMARY

Non-human animals comprising nucleic acid sequences encoding a DPP4protein that comprises a human sequence are provided.

Transgenic non-human animals comprising a DPP4 gene that is human inwhole or in part are provided.

Non-human animals that express human or humanized DPP4 protein areprovided.

Non-human animals having a replacement (in whole or in part) of theendogenous non-human animal Dpp4 gene are provided.

Non-human animals comprising a DPP4 humanization (in whole or in part)at an endogenous non-human Dpp4 locus are provided.

Non-human animals are provided that have a human or humanized DPP4 gene,wherein the non-human animals do not express endogenous Dpp4 protein,and wherein the non-human animals express human or humanized DPP4protein on the surface of immune cells, e.g., T cells, and/or on thesurface of cells in one or more tissues, including placenta, kidney,lung, liver, skeletal muscle, heart, brain and/or pancreas, at a levelsimilar to that of Dpp4 protein present on the surface of immune cells,e.g., T cells, and/or on the surface of cells in one or more tissues,including placenta, kidney, lung, liver, skeletal muscle, heart, brainand/or pancreas, of an age-matched non-human animal that expressesfunctional endogenous Dpp4 protein, but does not comprise thereplacement.

In one aspect, non-human animals comprising a human or humanized DPP4nucleic acid sequence are provided.

In one aspect, genetically modified non-human animals are provided thatcomprise a replacement at an endogenous Dpp4 locus of a gene encoding anendogenous Dpp4 gene encoding a human or humanized DPP4 protein.Rodents, e.g., mice or rats, are provided that comprise a replacement ofan endogenous Dpp4 gene, at an endogenous Dpp4 locus, with a human Dpp4gene. In one embodiment, the rodent is heterozygous for a replacement atan endogenous Dpp4 locus of an endogenous Dpp4 gene encoding a human orhumanized DPP4 protein. In one embodiment, the rodent is homozygous fora replacement at an endogenous Dpp4 locus of an endogenous Dpp4 geneencoding a human or humanized DPP4 protein. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided comprising a humanization of an endogenous rodent Dpp4 gene,wherein the humanization comprises a replacement at the endogenousrodent Dpp4 locus of a rodent gene encoding an exon of an Dpp4 gene witha nucleic acid sequence encoding at least one human DPP4 gene to form amodified DPP4 gene, wherein expression of the modified DPP4 gene isunder control of rodent regulatory elements at the endogenous rodentDpp4 locus.

In one embodiment, the rodent is heterozygous for the nucleic acidsequence encoding at least one exon of a human DPP4 gene to form amodified DPP4 gene. In one embodiment, the rodent is homozygous for thenucleic acid sequence encoding at least one exon of a human DPP4 gene toform a modified DPP4 gene.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the human DPP4 gene encoding a human or humanizedDPP4 protein comprises exon 2 through exon 26 of the human DPP4 gene.

In one embodiment, the humanized DPP4 protein comprises theextracellular domain of the human DPP4 protein.

In one embodiment, the humanized DPP4 protein comprises thetransmembrane domain and cytoplasmic domain of the mouse Dpp4 protein.

In one embodiment, the rodent is a mouse that is incapable of expressinga mouse Dpp4 protein.

In one embodiment, the rodent is a mouse wherein a contiguous genomicfragment of mouse Dpp4 sequence encoding exon 2 through exon 26 of mouseDpp4 is replaced with a contiguous genomic fragment of human DPP4sequence encoding exon 2 through exon 26 of human DPP4.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized DPP4 protein, wherein therodent that expresses a human or humanized DPP4 protein comprises anormal immune system, i.e., the number of immune cells, e.g., T cells,in the blood, plasma or serum of the rodent expressing human orhumanized DPP4 protein are similar to the number of immune cells, e.g.,T cells, in the blood, plasma or serum of a rodent that expressesfunctional endogenous Dpp4 protein. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one embodiment, the blood of the rodent that expresses a human orhumanized DPP4 protein has approximately the same number of immunecells, e.g., T cells, as a rodent that expresses a functional,endogenous Dpp4 protein, e.g., a wild-type mouse or rat. In oneembodiment, the rodent is a mouse. In one embodiment, the rodent is arat.

In one embodiment, the mouse expressing human or humanized DPP4 on thesurface of T cells has an amount of T cells present in the blood of atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the amount of Tcells present in the blood of an age-matched mouse that expressesfunctional endogenous Dpp4 protein, but does not comprise a replacementof an endogenous Dpp4 gene, at an endogenous mouse Dpp4 locus, with ahuman DPP4 gene.

In one embodiment, the mouse expressing human or humanized DPP4 proteinon the surface of T cells has an amount of T cells in the blood ofbetween about 20% and about 200%, between about 40% and about 160%, orbetween about 80% and about 120% of the amount of T cells present in theblood of an age-matched mouse that expresses functional endogenous Dpp4protein, but does not comprise a replacement of an endogenous Dpp4 gene,at an endogenous mouse Dpp4 locus, with a human DPP4 gene.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized DPP4 protein, wherein therodent expresses a human or humanized DPP4 protein on the surface ofimmune cells, e.g., T cells, and/or on the surface of cells in one ormore tissues, e.g., placenta, kidney, lung, liver, skeletal muscle,heart, brain and/or pancreas, of an age-matched rodent that expressesfunctional endogenous Dpp4 protein. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one embodiment, the immune cells, e.g., T cells, of the rodent thatexpresses a human or humanized DPP4 protein have approximately the samelevel of DPP4 protein on its surface as the immune cells, e.g., T cells,of a rodent that expresses a functional, endogenous Dpp4 protein, e.g.,a wild-type mouse or rat. In one embodiment, the rodent is a mouse. Inone embodiment, the rodent is a rat.

In one embodiment, the mouse expresses human or humanized DPP4 proteinon the surface of immune cells, e.g., T cells, at a level of at leastabout 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190% or 200% of the level of Dpp4 proteinon the surface of immune cells, e.g., T cells, of an age-matched mousethat expresses functional endogenous Dpp4 protein, but does not comprisea replacement of an endogenous Dpp4 gene, at an endogenous mouse Dpp4locus, with a human DPP4 gene.

In one embodiment, the mouse expresses human or humanized DPP4 proteinon the surface of immune cells, e.g., T cells, at a level of betweenabout 20% and about 200%, between about 40% and about 160%, or betweenabout 80% and about 120% of the level of mouse Dpp4 protein present onthe surface of immune cells, e.g., T cells, of an age-matched mouse thatexpresses functional endogenous Dpp4 protein, but does not comprise areplacement of an endogenous Dpp4 gene, at an endogenous mouse Dpp4locus, with a human DPP4 gene.

In one embodiment, the cells in one or more tissues, e.g., placenta,kidney; lung, liver, skeletal muscle, heart, brain and/or pancreas, ofthe rodent that expresses a human or humanized DPP4 protein haveapproximately the same level of DPP4 protein on its surface as the cellsin one or more tissues, e.g., placenta, kidney, lung, liver, skeletalmuscle, heart, brain and/or pancreas, of a rodent that expresses afunctional, endogenous Dpp4 protein, e.g., a wild-type mouse or rat. Inone embodiment, the rodent is a mouse. In one embodiment, the rodent isa rat.

In one embodiment, the mouse expresses human or humanized DPP4 proteinon the surface of cells in one or more tissues, e.g., placenta, kidney,lung, liver, skeletal muscle, heart, brain and/or pancreas, at a levelof at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the level ofDpp4 protein on the surface of cells in one or more tissues, e.g.,placenta, kidney, lung, liver, skeletal muscle, heart, brain and/orpancreas, of an age-matched mouse that expresses functional endogenousDpp4 protein, but does not comprise a replacement of an endogenous Dpp4gene, at an endogenous mouse Dpp4 locus, with a human DPP4 gene.

In one embodiment, the mouse expresses human or humanized DPP4 proteinon the surface of cells in one or more tissues, e.g., placenta, kidney,lung, liver, skeletal muscle, heart, brain and/or pancreas, at a levelof between about 20% and about 200%, between about 40% and about 160%,or between about 80% and about 120% of the level of mouse Dpp4 proteinpresent on the surface of cells in one or more tissues, e.g., placenta,kidney, lung, liver, skeletal muscle, heart, brain and/or pancreas, ofan age-matched mouse that expresses functional endogenous Dpp4 protein,but does not comprise a replacement of an endogenous Dpp4 gene, at anendogenous mouse Dpp4 locus, with a human DPP4 gene.

In one aspect, a genetically modified rodent is provided, comprising ahumanized DPP4 gene comprising a replacement of rodent Dpp4extracellular domain-encoding sequence with human DPP4 extracellulardomain-coding sequence, wherein the humanized DPP4 gene comprises arodent Dpp4 transmembrane sequence and a rodent Dpp4 cytoplasmicsequence, wherein the humanized DPP4 gene is under control of endogenousrodent Dpp4 regulatory elements at the endogenous Dpp4 locus.

In one embodiment, the rodent is heterozygous for the humanized DPP4gene. In one embodiment, the rodent is homozygous for the humanized DPP4gene.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the mouse is incapable of expressing a mouse Dpp4protein.

In one embodiment, the rodent regulatory elements or sequences at theendogenous rodent Dpp4 locus are from a mouse or a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent Dpp4locus are from a mouse or a rat.

In one aspect, a non-human animal, e.g., a rodent, e.g., a mouse or rat,is provided that expresses human or humanized DPP4 protein, wherein thenon-human animal expresses human or humanized DPP4 protein from anendogenous non-human Dpp4 locus. In an embodiment, the non-human animalis a rodent. In an embodiment, the rodent is a mouse. In an embodiment,the rodent is a rat. In one embodiment, the rodent is heterozygous forthe endogenous non-human Dpp4 locus expressing a human or humanized DPP4protein. In one embodiment, the rodent is homozygous for the endogenousnon-human Dpp4 locus expressing a human or humanized DPP4 protein.

In one aspect, a genetically modified mouse is provided that expresseshuman or humanized DPP4 protein from an endogenous mouse Dpp4 locus,wherein the endogenous mouse Dpp4 gene has been replaced, in whole or inpart, with a human DPP4 gene.

In one embodiment, about 78.8 kb at the endogenous mouse Dpp4 locus,including exon 2 through the stop codon in exon 26, is deleted andreplaced with about 81.8 kb of human DPP4 gene sequence comprising exon2 through exon 26 and a portion of the 3′ untranslated sequence of thehuman DPP4 gene. In a specific embodiment, the human DPP4 gene comprisesexon 2 through exon 26 and a portion of the 3′ untranslated sequence ofthe human DPP4 gene of human BAC RP11-68L22. In a specific embodiment,the DPP4 gene comprises mouse Dpp4 gene 5′ regulatory elements, mouseDpp4 exon 1, including the first two amino acids, Met and Lys, of themouse Dpp4 protein, and mouse Dpp4 3′ regulatory elements (e.g., 3′untranslated region), and human DPP4 gene exon 2 through exon 26, i.e.,the human DPP4 protein coding sequences, except for the first two aminoacids, which are derived from mouse Dpp4 exon 1.

In one aspect, a genetically modified mouse is provided that comprises anucleotide sequence encoding a human or humanized DPP4 protein, whereinthe nucleotide sequence encoding the human or humanized DPP4 proteinreplaces, in whole or in part, an endogenous nucleotide sequenceencoding an endogenous mouse Dpp4 protein.

In one embodiment, the mouse is heterozygous for the nucleotide sequenceencoding a human or humanized DPP4 protein. In one embodiment, the mouseis homozygous for the nucleotide sequence encoding a human or humanizedDPP4 protein.

In one aspect, a method is provided for making a humanized DPP4 rodent,comprising replacing a rodent Dpp4 gene sequence encoding rodent Dpp4protein with a human DPP4 gene sequence comprising one or more exons ofthe human DPP4 gene sequence encoding human or humanized DPP4 protein,wherein the replacement is at an endogenous rodent Dpp4 locus and thehuman DPP4 gene sequence comprising one or more exons of the human DPP4gene sequence encoding human or humanized DPP4 protein is operablylinked to rodent regulatory elements or sequences at the endogenousrodent Dpp4 locus.

In one embodiment, the rodent is heterozygous for the nucleotidesequence encoding a human or humanized DPP4 protein. In one embodiment,the rodent is homozygous for the nucleotide sequence encoding a human orhumanized DPP4 protein.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the rodent regulatory elements or sequences arederived from a mouse. In one embodiment, the rodent regulatory elementsor sequences are derived from a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent Dpp4locus. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the human DPP4 gene sequence replacing the rodentDpp4 gene sequence comprises at least one exon of the human DPP4 genesequence. In other embodiments, the human DPP4 gene sequence replacingthe rodent Dpp4 gene sequence comprises at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, or at least 25 exons of thehuman DPP4 gene sequence. In one embodiment, the human DPP4 genesequence replacing the rodent Dpp4 gene sequence comprises all 26 exonsof the human DPP4 gene sequence. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one embodiment, the human or humanized DPP4 gene sequence replacingthe rodent Dpp4 gene sequence encodes a protein that is about 85%, 90%,95%, 96%, 97%, 98%, or about 99% identical to a human DPP4

In one embodiment, the human or humanized DPP4 gene sequence replacingthe rodent Dpp4 gene sequence comprises at least one exon of the humanDPP4 gene sequence encoding the extracellular domain of the human DPP4protein. In other embodiments, the human DPP4 gene sequence replacingthe rodent Dpp4 gene sequence comprises at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, or at least 23 exons of the human DPP4 gene sequenceencoding the extracellular domain of the human DPP4 protein. In oneembodiment, the human DPP4 gene sequence replacing the rodent Dpp4 genesequence comprises all 24 exons of the human DPP4 gene sequence encodingthe extracellular domain of the human DPP4 protein. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the human or humanized DPP4 gene sequence replacingthe rodent Dpp4 gene sequence encodes an extracellular domain of theDPP4 protein that is about 85%, 90%, 95%, 96%, 97%, 98%, or about 99%identical to the extracellular domain of a human DPP4 protein.

In one embodiment, the replacement is at an endogenous rodent Dpp4 locusand the human DPP4 gene sequence comprising one or more exons of thehuman DPP4 gene sequence encoding human or humanized DPP4 protein isoperably linked to endogenous rodent regulatory elements or sequences atthe endogenous rodent Dpp4 locus.

In one aspect, a method is provided for making a humanized DPP4 mouse,comprising replacing a mouse Dpp4 gene sequence encoding mouse Dpp4protein with a human DPP4 gene sequence encoding human or humanized DPP4protein.

In one embodiment, the replacement is at an endogenous mouse Dpp4 locus,and the human DPP4 gene encoding human or humanized DPP4 protein isoperably linked to mouse regulatory elements or sequences at theendogenous mouse Dpp4 locus.

In one embodiment, the replacement is at an endogenous mouse Dpp4 locus,and the human DPP4 gene encoding human or humanized DPP4 protein isoperably linked to endogenous mouse regulatory elements or sequences atthe endogenous mouse Dpp4 locus.

In various aspects, the genetically modified non-human animals, e.g.,rodents, e.g., mice or rats, described herein comprise the geneticmodifications in their germ-line.

In one aspect, a non-human animal, e.g., rodent, e.g., a mouse or rat,embryo comprising a genetic modification as described herein isprovided.

In one aspect, a non-human animal, e.g., rodent, e.g. a mouse or rat,host embryo is provided that comprises a donor cell that comprises agenetic modification as described herein.

In one aspect, a pluripotent or totipotent non-human animal, e.g.,rodent, e.g., mouse or rat, cell comprising a genetic modification asdescribed herein is provided. In one embodiment, the cell is a rodentcell. In one embodiment, the cell is a mouse cell. In one embodiment,the cell is a rodent embryonic stem (ES) cell. In one embodiment, thecell is a mouse ES cell.

In one aspect, a non-human animal, e.g., rodent, e.g., mouse or rat, eggis provided, wherein the non-human animal egg comprises an ectopicnon-human animal chromosome, wherein the ectopic non-human animalchromosome comprises a genetic modification as described herein. In oneembodiment, the non-human animal is a rodent. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, the mouse embryo, egg, or cell that is geneticallymodified to comprise a human DPP4 gene is of a mouse that is of a C57BLstrain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/61, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn,C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129strain selected from the group consisting of a strain that is 129P1,129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4,129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al. (1999) Revised nomenclature for strain 129mice, Mammalian Genome 10:836, see also, Auerbach et al (2000)Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-DerivedMouse Embryonic Stem Cell Lines). In a specific embodiment, thegenetically modified mouse is a mix of an aforementioned 129 strain andan aforementioned C57BL/6 strain. In another specific embodiment, themouse is a mix of aforementioned 129 strains, or a mix of aforementionedBL/6 strains. In a specific embodiment, the 129 strain of the mix is a129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALBstrain, e.g., BALB/c strain. In yet another embodiment, the mouse is amix of a BALB strain and another aforementioned strain. In oneembodiment, the mouse is Swiss or Swiss Webster mouse.

In various aspects, the non-human animals comprising a human orhumanized DPP4 nucleic acid sequence are selected from mammals andbirds. In one embodiment, the non-human animals are mammals. In oneembodiment, the mammals are rodents. In one embodiment, the rodents aremice or rats.

In one aspect, a rodent is provided that comprises a nucleic acidsequence comprising a human DPP4 gene or fragment thereof, where thehuman DPP4 gene or fragment thereof comprises at least one exon of thehuman DPP4 gene, and where the human DPP4 gene or fragment thereofencodes a human or humanized DPP4 protein.

In one embodiment, the human DPP4 gene or fragment thereof comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18,20, 21, 22, 23, 24, or 25 exons of the human DPP4 gene.

In one embodiment, the human DPP4 gene or fragment thereof comprises all26 exons of the human DPP4 gene.

In one embodiment, the nucleic acid sequence further comprises a 5′flanking region of the human DPP4 gene. In one embodiment, the humanDPP4 gene or fragment thereof is operably linked to the 5′ flankingregion of the human DPP4 gene. In one embodiment, the 5′ flanking regionof the human DPP4 gene comprises at least 1 kb in length (e.g., at least1, 2, 3, 4, 5, 10, 20, 30, 40, 50 kb, or greater, in length). In oneembodiment, the 5′ flanking region of the human DPP4 gene comprises atleast 10 kb in length. In one embodiment, the 5′ flanking region of thehuman DPP4 gene comprises at least 40 kb in length.

In one embodiment, expression of the human DPP4 gene or fragment thereofis under control of the 5′ flanking region of the human DPP4 gene.

In one embodiment, the human or humanized DPP4 protein comprises theamino acid sequence of SEQ ID NO: 24 or a fragment thereof.

In one embodiment, the rodent expresses the human or humanized DPP4protein on the surface of T cells in a level that is at least about 20%(e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, or greater) of the level of rodent Dpp4 protein present on thesurface of T cells of an age-matched rodent that expresses functionalendogenous rodent Dpp4 protein but that does not comprise the human DPP4gene or fragment thereof.

In one embodiment, the rodent expresses the human or humanized DPP4protein on the surface of cells in one or more tissues selected from thegroup consisting of placenta, kidney, lung, liver, skeletal muscle,heart, brain, and pancreas, in a level that is at least about 20% (e.g.,at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, orgreater) of the level of rodent Dpp4 protein present on the surface ofone or more tissues of an age-matched rodent that expresses functionalendogenous rodent Dpp4 protein but that does not comprise the human DPP4gene or fragment thereof.

In one embodiment, the rodent expresses functional endogenous rodentDpp4 protein.

In one embodiment, the rodent is a mouse or a rat.

In one aspect, provided herein is a method for making a humanizedtransgenic rodent, comprising integrating a nucleic acid sequencecomprising one or more exons of a human DPP4 gene sequence into achromosome of a rodent, where the one or more exons of the human DPP4gene sequence encodes a human or humanized DPP4 protein.

In one embodiment, the human DPP4 gene or fragment thereof comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18,20, 21, 22, 23, 24, or 25 exons of the human DPP4 gene.

In one embodiment, the human DPP4 gene or fragment thereof comprises all26 exons of the human DPP4 gene.

In one embodiment, the nucleic acid sequence further comprises a 5′flanking region of the human DPP4 gene.

In one embodiment, the human DPP4 gene sequence is operably linked tothe 5′ flanking region of the human DPP4 gene.

In one embodiment, the human or humanized DPP4 protein comprises theamino acid sequence of SEQ ID NO: 24 or a fragment thereof.

In one embodiment, the rodent is a mouse or a rat. In further aspects,methods for determining the in vivo therapeutic efficacy of ahuman-specific DPP4 antagonist in any of the humanized DPP4 rodentsdescribed herein are provided, the method comprising administering tothe rodent a DPP4 antagonist, wherein the rodent is infected with MiddleEast respiratory syndrome coronavirus (MERS-CoV); and determining ifwhether the DPP4 antagonist treats or prevents one or more symptoms ofMERS-CoV infection compared to control rodents infected with MERS-CoVwho have not been administered the DPP4 antagonist.

In one embodiment, the DPP4 antagonist is selected from the groupconsisting of small molecules, peptides and antibodies.

In one embodiment, the DPP4 antagonist is an antibody to a MERS-CoVprotein.

In one embodiment, the MERS-CoV protein is MERS-CoV spike protein.

In one embodiment, the rodent is infected with one or more strains ofMERS-CoV selected from the group consisting of Al-Hasa_1, Al-Hasa_2,Al-Hasa_3, Al-Hasa_4, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17,Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-Hasa_25, Buraidah_1, EMC/2012,FRA/UAE, Hafr-Al-Batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1,Jordan-N3/2012, Munich, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_14, Taif_1,Wadi-Ad-Dawasir_1, Riyadh_9, KFU-HKU 1, KFU-HKU 13, Qatar3, Qatar4,England 1, England-Qatar/2012, Bisha_1, Riyadh_1, and Riyadh_2.

In one embodiment, the antagonist is administered before MERS-CoVinfection. In one embodiment, the antagonist is administered afterMERS-CoV infection.

In one embodiment, the antagonist is administered simultaneously withMERS-CoV infection.

In one embodiment, the symptom of MERS-CoV infection is viral titer orRNA level.

In one embodiment, the viral titer or RNA level is assessed by one ormore methods selected from the group consisting of qPCR, Northern Blot,plaque assay, and in situ hybridization,

In one embodiment, the symptom of MERS-CoV infection is lunginflammation.

In one embodiment, the lung inflammation is assessed histochemically

In one embodiment, the symptom of MERS-CoV infection is weight loss.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B provide illustrations, not to scale, of the strategyfor humanization of the Dpp4 locus. FIG. 1A is a schematic showing that78.8 kb of the mouse Dpp4 gene (top) spanning exon 2 through the stopcodon in exon 26 are deleted and replaced with 81.7 kb of the human DPP4gene (bottom) spanning exon 2 through exon 26 and a portion of the 3′untranslated region, as indicated. FIG. 1B is a schematic showing thatthe humanized DPP4 mouse comprises (i) the mouse Dpp4 gene 5′ flankingregion, including the regulatory sequences, e.g., promoter andtranscription start site, and exon 1, including the initiation ATGcodon, (ii) the human DPP4 gene spanning exon 2 through exon 26,including the Stop codon, and a portion of the 3′untranslated region,including the loxP site, and (iii) the mouse Dpp4 gene 3′ untranslatedregion starting from just 3′ to the Stop codon, as indicated.

FIG. 2 shows the amino acid sequence (SEQ ID NO:17) of the humanizedDPP4 protein expressed in the humanized DPP4 mice.

FIG. 3 shows the results of real-time PCR performed on RNA obtained fromlung tissue from either mock (PBS)-infected, or MERS-CoV (Jordanstrain)-infected, F0 humanized DPP4 mice 4 days after infection.

FIG. 4 shows the HU staining of airway (10× and 40× magnification) andalveoli (40× magnification) from the lungs of either mock(PBS)-infected, or MERS-CoV (Jordan strain)-infected, F0 humanized DPP4mice 4 days after infection.

FIG. 5 is a sequence alignment of the mouse Dpp4 (mDpp4) amino acidsequence (SEQ ID NO: 25) with the human DPP4 (hDPP4) protein encoded bythe transgenic MAID 7326/7327 mice (SEQ ID NO: 26). Non-homologousresidues that differ between the sequences are underlined, homologousresidues that differ between the sequences are bolded and italicized,and gaps are indicated by hyphens. Residues that are identical betweenthe two sequences are shown in unformatted text.

FIG. 6 is a table displaying gene, sequence, and chromosomal informationfor human DPP4.

FIG. 7 is a schematic showing the coverage of the human DPP4 gene andflanking genomic sequences for each of the BACs, BAC RP11-345J9 and BACRP11-68L22. The locations within the human DPP4 gene and promoterregions at which the human TaqMan™ primer-probe sets anneal are alsoshown (7333hTU for the upstream set and 7333hTD for the downstream set).

FIG. 8 is a table displaying the primer-probe sets used for the humanTaqMan™ gain of allele assays, where 7333 hTU refers to the upstream setand 7333 hTD refers to the downstream set.

FIG. 9 is the amino acid sequence of the humanized DPP4 protein encodedby the transgenic MAID 7333 and 7334 mice (SEQ ID NO: 24).

FIG. 10A is a bar graph showing quantitative PCR measurements ofMERS-CoV genome (UpE RNA) in infected mice 2 and 4 days post-infection(dpi). FIG. 10B is a bar graph showing quantitative PCR measurements ofMERS-CoV mRNA transcript (leader RNA) in infected mice on 2 dpi and 4dpi. FIG. 10C is a bar graph quantifying MERS-CoV viral titer ofinfected mouse lung at 2 dpi and 4 dpi. MERS-CoV levels in homogenizedmouse lung were quantified by 50% Tissue Culture Infective Dose (TCID50)assay and expressed as plaque forming units (pfu) per mL. FIG. 10D is apanel of histological images, stained with Hematoxylin and Eosin, oflungs from MERS-CoV infected mice. Airway (10×), vasculature (10×) andinterstitium (40×) are shown for PBS, 2 dpi, and 4 dpi mice.

FIG. 11A is a bar graph showing quantitative PCR measurements ofMERS-CoV genome (UpE RNA) from lungs of mice pre-treated withanti-MERS-CoV spike protein antibodies (Ab 2 or Ab 4) before viralinfection. FIG. 11B is a bar graph showing quantitative PCR measurementsof MERS-CoV mRNA transcript (leader RNA) from lungs of mice pre-treatedwith anti-MERS-CoV spike protein antibodies before viral infection. RNAwas quantified using primers directed against the MERS-CoV genome andcompared to hIgG 1 isotype control treated mice. All samples werecompared to hIgG 1 control set at 100%. FIG. 11C is a bar graph showingthe viral titer in lungs of mice pre-treated with anti-MERS-CoV spikeprotein antibodies before viral infection. Viral titer was quantitatedby plaque assay and reported as pfu/mL.

FIG. 12A is a panel of histological images of lungs of MERS-CoV infectedB6/hDPP4 mice with anti-MERS-CoV spike protein antibody (Ab 2 or Ab 4)pre-treatment. Hematoxylin and Eosin stained sections of mouse lungshowing airway, vasculature and interstitium of a representative mousefrom each group. FIG. 12B is a bar graph showing the histologicalscoring of the mouse lungs shown in FIG. 12A. The scores were theaverage scores of all mice in each experimental group and time point.

FIG. 13A is a bar graph showing quantitative PCR measurements ofMERS-CoV genome (UpE RNA) from infected lungs. Effects of anti-MERS-CoVspike protein antibodies (Ab 2 or Ab 4) injected one day before or oneday after viral infection were compared. FIG. 13B is a bar graph showingquantitative PCR measurements of MERS-CoV mRNA transcript (leader RNA)from infected lungs. Effects of anti-MERS-CoV spike protein antibodies(Ab 2 or Ab 4) injected one day before or one day after viral infectionwere compared. For FIG. 13A and FIG. 13B, RNA was quantified usingprimers directed against the MERS-CoV genome and compared to hIgG 1isotype control treated mice. All samples were compared to hIgG 1control set at 100%. FIG. 13C is a bar graph showing the viral titer inlungs of mice treated with anti-MERS-Co V spike protein antibodies (Ab 2or Ab 4) after viral infection. Viral titer was quantitated by plaqueassay and reported as PFU/mL lung. Effects of antibodies injected oneday before or one day after viral infection were compared.

FIG. 14A is a panel of histological images of lungs from MERS-CoVinfected B6/hDPP4 mice with anti-MERS-CoV spike protein Ab 2 antibodytreatment at 1 day post-infection. Hematoxylin and Eosin stainedsections of mouse lung show airway, vasculature, and interstitium of arepresentative mouse from each group. FIG. 14B is a bar graph showingthe histological scoring of the mouse lungs of FIG. 14A.

FIG. 15 depicts a dose-response study of weight as a function of timepost-infection with MERS-CoV in humanized DPP4 mice. 4-5 mice are usedper group.

FIG. 16 depicts a dose-response study of weight as a function of timepost-infection with MERS-CoV (1×10⁶ pfu/mouse; 4-5 mice per group) inheterozygotic and homozygotic humanized DPP4 mice.

FIG. 17 depicts pathology (inflammation) as seen by histologicalexamination at day 7 in a humanized DPP4 mouse exposed to a high dose ofvirus (1×10⁶ pfu/mouse) versus PBS-treated controls.

DETAILED DESCRIPTION DPP4 Gene and Protein

The DPP4 gene encodes the type II transmembrane protein, tripeptidylpeptidase IV (DPP4) (also known as CD26, adenosine deaminase complexingprotein-2 (ADCP2), adenosine deaminase binding protein (ADABP), andTP103), which has serine exopeptidase activity, and which plays animportant role in the activation of T cells and in intracellular signaltransduction cascades in several other cell types.

Human DPP4.

NCBI Gene ID: 1803; Primary source: HGNC:3009; RefSeq transcript:NM_001935.3; UniProt ID: P27487; Genomic assembly: GRCh38; Location:chr2:162,848,755-162,931,052-strand. (See FIG. 6).

The human DPP4 gene is located on chromosome 2, at 2q24.3. The humanDPP4 gene has 26 exons and encodes a type II transmembrane polypeptideof 766 amino acids in length, including an N-terminal 6 amino acidcytoplasmic domain, a 22 amino acid transmembrane domain, and aC-terminal 738 amino acid extracellular domain. The extracellular domain(i.e., ectodomain) of the human DPP4 protein is encoded by coding exons3 through 26 of the human DPP4 gene.

Mouse Dpp44.

NCBI Gene ID: 13482; Primary source: MGI:94919; RefSeq transcript:NM_010074.3; UniProt ID: P28843; Genomic assembly: GRCm38; Location:chr2:62,330,073-62,412,591-strand.

The mouse Dpp4 gene is located on chromosome 2, at 2 35.85 cM. The mouseDpp4 gene has 26 exons and encodes a type II transmembrane polypeptideof 760 amino acids in length, including an N-terminal 6 amino acidcytoplasmic domain, a 22 amino acid transmembrane domain, and aC-terminal 732 amino acid extracellular domain. The extracellular domain(i.e., ectodomain) of the mouse Dpp4 protein is encoded by coding exons3 through 26 of the mouse Dpp4 gene.

Species Specificity of DPP4 Protein

As discussed below in Example 2, the human, but not the mouse, DPP4protein is a functional receptor for the Middle East respiratorysyndrome coronavirus (MERS-CoV) infection.

Candidate therapeutic molecules that target the human DPP4 protein in aspecies-specific manner, or target molecules, such as MERS-CoV, whichinteract with the human DPP4 protein in a species-specific manner, aretypically evaluated for pharmacokinetics (PK) and pharmacodynamics (PK)in non-human animals, e.g., rodents, e.g., mice or rats. Suchtherapeutic molecules are also tested for in vivo therapeutic efficacyin non-human animal, e.g., rodent, e.g., mouse or rat, models of humandiseases, disorders and conditions in which DPP4 plays a role.

However, therapeutic molecules that are specific for the human DPP4protein, e.g., human-specific DPP4 inhibitors, cannot be adequatelyevaluated for PD and/or in vivo therapeutic efficacy in rodents, inparticular mice, because the targets of these therapeutic molecules aremissing.

Moreover, therapeutic molecules that are specific for targets thatspecifically interact with human DPP4 protein, e.g., human DPP4-specificMERS-CoV, cannot be adequately evaluated for in vivo therapeuticefficacy in rodents, in particular mice, because the targets (e.g.,receptor, interaction partner) of these therapeutic target molecules aremissing.

Accordingly, in various embodiments, to assess the PD and/or in vivotherapeutic efficacy of a human-specific DPP4 protein antagonist orinhibitor in non-human animals, e.g., rodents, e.g., mice or rats, it isdesirable to replace the endogenous Dpp4 protein with human or humanizedDPP4 protein. In various embodiments, to assess the in vivo therapeuticefficacy of small molecules, peptides or biologic antagonists orinhibitors of a target molecule that specifically interacts with a humanDPP4 protein in non-human animals, e.g., rodents, e.g., mice or rats, itis desirable to replace the endogenous Dpp4 protein with human orhumanized DPP4 protein.

Further, in various embodiments, in order to avoid potential problems ofthe over- or under-expression of the human or humanized DPP4 protein,and/or the inappropriate expression of the human or humanized DPP4protein in cells or tissues in which the endogenous Dpp4 protein is notnormally expressed, it is desirable to insert the human DPP4 gene, inwhole or in part, into the genome of the non-human animals, e.g.,rodents, e.g., mice or rats, at the endogenous Dpp4 gene loci, and toexpress the human or humanized DPP4 protein in non-human animals, e.g.,rodents, e.g., mice or rats, under the control, at least in part, of theendogenous Dpp4 regulatory elements.

In some embodiments, targeted replacement of the endogenous, e.g., mouseor rat, Dpp4 gene by the human DPP4 gene or fragment thereof isdesirable.

In other embodiments, the human DPP4 gene or fragment thereof israndomly inserted into the rodent, e.g., mouse or rat, genome instead ofreplacing the endogenous Dpp4 gene with a human DPP4 gene or fragmentthereof. In some embodiments, in rodents, e.g., mice or rats, in whichthe human DPP4 gene or fragment thereof has been randomly inserted intothe genome, expression of endogenous rodent Dpp4 is retained.

Provided herein are non-human animals, e.g., rodents, e.g., mice orrats, that comprise a human DPP4 gene or fragment thereof either at(i.e., replacing) the endogenous Dpp4 locus, or at one or more otherloci. Also provided herein are non-human animals, e.g., rodents, e.g.,mice or rats, that comprise a human DPP4 gene or fragment thereof bothat (i.e., replacing) the endogenous Dpp4 locus, and at an additionallocus/loci.

In some embodiments, a fragment of a human DPP4 gene contains 200kilobases (kb) or fewer nucleotides, e.g., 180, 160, 140, 120, 100, 80,70, 60, 50, 40, 30, 20, 10, 5, 2.5, 1 kb or fewer nucleotides, e.g.,1000, 800, 600, 400, 200, or fewer nucleotides.

Generation of Cells and Non-Human Animals with Human DPP4

For targeted replacement of an endogenous non-human Dpp4 gene orfragment with a human DPP4 gene or fragment, a targeting construct isgenerated. See, e.g., Valenzuela et al. Nature Biotech,21.6(2003):652-659; U.S. Pat. Nos. 6,586,251; and 8,759,105. Forexample, a targeting construct comprises homology arms flanking areplacement human DPP4 gene or fragment thereof.

In some embodiments, the replacement human DPP4 gene or fragment thereofcomprises the entire human DPP4 gene. In other embodiments, thereplacement human DPP4 gene or fragment thereof comprises a portion ofthe human DPP4 gene. For example, the replacement human DPP4 gene orfragment thereof comprises one or more exons of human DPP4 gene, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, or 26 exons of the human DPP4 gene. For example, thereplacement human DPP4 gene or fragment thereof comprises the exons 1-26of the human DPP4 gene. In other embodiments, the replacement human DPP4gene or fragment thereof comprises exons 2-26 of the human DPP4 gene.For example, the replacement human DPP4 gene or fragment thereofcomprises intron 1 upstream of exon 2 through exon 26 of the human DPP4gene. In some embodiments, the replacement human DPP4 gene or fragmentthereof further comprises a human regulatory element(s), e.g., a portionof the human 3′ untranslated region (UTR) downstream of the human DPP4gene, for example at least 1 kb of downstream region (e.g., at least 1,2, 3, 4, 5, 10, 20, 30, 40 Kb, or greater), and/or a human promoter orenhancer region upstream of the human DPP4 gene, for example, at least10 kb of upstream region (e.g., at least 10, 20, 30, 40, 50 Kb, orgreater).

Homology arms are sequences that are homologous to endogenouschromosomal nucleic acid sequences flanking the desired geneticmodification/replacement, e.g., flanking the endogenous Dpp4 gene orfragment that is to be replaced. Homologous nucleic acid sequences canbe two or more nucleic acid sequences that are either identical orsimilar enough that they are able to hybridize to each other or undergointermolecular exchange. Due to the homology between the homology armsand the corresponding endogenous sequence, the homology arms direct thetargeting construct to a specific chromosomal location within thegenome, e.g., the endogenous Dpp4 gene locus. See, e.g., Valenzuela etal. Nature Biotech, 21.6(2003):652-659; U.S. Pat. Nos. 6,586,251; and8,759,105.

Optionally, the targeting construct further comprises a selectionmarker, e.g., in between the two homology arms. Exemplary selectionmarkers include antibiotic resistance markers (e.g., neomycin orkanamycin) and fluorescent proteins. In some embodiments, the selectionmarker is foxed, i.e., flanked by two loxP sites. The floxed selectionmarker can be removed by the addition of Cre recombinase, whichcatalyzes the excision of the floxed segment, e.g., including theselection marker.

Vector/Constructs

The transgenic non-human animals (e.g., rodents, e.g., mice or rats) ofthe invention can be made by using various vectors and/or constructs. Insome embodiments, the targeting construct is in the form of a circularpiece of double-stranded DNA, e.g., a bacterial artificial chromosome(BAC), plasmid, or P1-derived artificial chromosome (PAC).

To generate a non-human cell comprising a targeted replacement of theendogenous Dpp4 locus, a targeting construct containing a human DPP4gene or fragment described herein is introduced into a non-human (e.g.,rodent, e.g., mouse or rat) cell, e.g., embryonic stem (ES) cell.

To generate a non-human cell comprising a human DPP4 gene or fragmentrandomly inserted into the genome, a circular DNA construct, e.g., BAC,containing a human DPP4 gene or fragment thereof, is introduced into anon-human (e.g., rodent, e.g., mouse or rat) cell, e.g., ES cell. Insome cases, the circular DNA construct, e.g., BAC, further contains ahuman DPP4 regulatory element, e.g., a human promoter or enhancer regionupstream and/or downstream of human DPP4 gene. For example, the circularDNA construct contains at least 10 kb (e.g., at least 10, 20, 30, 40, 50kb or greater) of promoter/enhancer region upstream of the ATG startcodon of the human DPP4 gene. In addition or alternatively, the circularDNA construct contains at least 1 kb (e.g., at least 1, 2, 3, 4, 5, 10,20, 30, 40 kb or greater) of untranslated region downstream of the humanDPP4 gene. For example, the human DPP4 gene or fragment is operablylinked to the human DPP4 regulatory element.

In some embodiments, the human DPP4 gene or fragment thereof in thecircular DNA construct (e.g., BAC) comprises the entire human DPP4 gene.In other embodiments, the human DPP4 gene or fragment thereof comprisesa portion of the human DPP4 gene. For example, the human DPP4 gene orfragment thereof comprises one or more exons of human DPP4 gene, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, or 26 exons of the human DPP4 gene. For example, thehuman DPP4 gene or fragment thereof comprises the exons 1-26 of thehuman DPP4 gene. In other embodiments, the human DPP4 gene or fragmentthereof comprises exons 2-26 of the human DPP4 gene. For example, thehuman DPP4 gene or fragment thereof comprises intron 1 upstream of exon2 through exon 26 of the human DPP4 gene.

For example, the introduction step into the cell is done byelectroporation or lipid-mediated transfection.

Optionally, the circular DNA construct, e.g., BAC, is linearized beforeintroduction into the cell. For example, linearization is performed withrare-cutting restriction enzymes, e.g., SgrDI, SfiI, NotI, PacI, orSwaI.

In cases in which the targeting construct comprises an antibioticselection marker (e.g., neomycin), cells that have taken up thetargeting construct are optionally selected in neomycin/G418-containingmedia. Cells that survive and/or proliferate in neomycin/G418-containingmedia are selected and positive for the targeting construct.

In some embodiments, the cell population is screened for those cellsthat have incorporated into their genome a human DPP4 gene or fragmentthereof, e.g., randomly inserted into the genome or targeted (e.g., bythe targeting construct) to the endogenous Dpp4 locus.

Methods for screening include quantitative PCR and fluorescence in situhybridization. See, e.g., U.S. Pat. No. 6,586,251 B2 and U.S. Pat. No.8,759,105 B2. For example, methods of screening include detecting forthe presence of a human DPP4 gene or fragment. In some embodiments,methods of screening include detecting for a loss of copy number ofendogenous Dpp4 gene or fragment and/or gain of copy number of humanDPP4 gene or fragment. Exemplary methods of screening are described inExamples 1 and 3.

In some embodiments in which the targeting construct comprises a floxedselection marker, correctly targeted cells are optionally furtherelectroporated with a Cre-expressing vector, e.g., transientlyexpressing Cre recombinase, to remove the floxed selection marker.

To generate transgenic animals, positive ES cell clones, e.g., withoutfloxed selection marker, containing a human DPP4 gene or fragmentthereof, are introduced into a rodent embryo, e.g., a mouse or ratembryo, such as an 8-cell stage mouse embryo. For example, theintroduction step is done by blastocyst injection technology,aggregation techniques, nuclear transfer and cloning, and/or theVelociMouse® method. See, e.g., U.S. Pat. No. 8,759,105 B2, U.S. Pat.Nos. 7,294,754, 7,576,259, and 7,659,442. For example, an ES cell cloneis a subpopulation of cells derived from a single cell of the ES cellpopulation following introduction of DNA and subsequent selection.

In some cases, DNA from transgenic non-human animals are screened insimilar ways as described above to confirm transmittance of the humanDPP4 gene/fragment through the germline.

In some embodiments, the humanized DPP4 rodents described herein areheterozygous for the human DPP4 allele. As such, these rodents have onehuman DPP4 allele and one wild-type rodent DPP4 allele. In otherembodiments, the humanized DPP4 rodents are homozygous for the humanDPP4 allele.

Uses for Humanized DPP4 Rodents

Humanized DPP4 rodents, e.g., mice or rats, are useful to evaluate thepharmacodynamics (PD) of human-specific DPP4 antagonists, e.g., smallmolecule, peptide or biologic inhibitors, useful for the treatment ofhyperglycemia.

Pharmacokinetics (PK) and PD assays in humanized DPP4 rodents, e.g, miceor rats, are performed according to standard procedures known in theart.

Humanized DPP4 rodents, e.g., mice or rats, are useful to evaluate thein vivo therapeutic efficacy of human-specific DPP4 antagonists, e.g.,small molecule, peptide or biologic inhibitors, in the treatment ofhyperglycemia.

Humanized DPP4 rodents, e.g., mice or rats, are useful to test the invivo therapeutic efficacy of antagonists, e.g., small molecule, peptideor biologic inhibitors, e.g., neutralizing antibodies, that are specificfor target molecules, e.g., MERS-CoV (e.g., spike protein (S) ofMERS-CoV, e.g., receptor binding domain of the spike protein ofMERS-CoV), which specifically interact with human DPP4, in the treatmentor prevention (or prophylaxis) of MERS-CoV infection. In someembodiments, rodents that are heterozygous for the human DPP4 allele areused to test the in vivo therapeutic efficacy of one or more antagonistsin the treatment or prevention (or prophylaxis) of ME RS-Co V infection.In other embodiments, DPP4 rodents that are homozygous for the humanDPP4 allele are used to test the in vivo therapeutic efficacy of one ormore antagonists in the treatment or prevention (or prophylaxis) ofMERS-CoV infection.

Exemplary MERS-CoV strains include MERS-CoV Jordan strain (GenBankaccession no. KC776174.1, MERS-CoV-Hu/Jordan-N3/2012) and MERS-CoVEMC/2012 strain (GenBank accession no. JX869059.2). In some embodiments,a MERS-CoV virus described herein comprises a MERS-CoV clinical isolate.In other embodiments, a MERS-CoV virus described herein comprises astrain comprising the same spike protein receptor binding domain (RBD)sequence as a clinical isolate described herein. Exemplary clinicalisolates are shown in the table below. The table shows the amino acidsequence variation within the receptor binding domain (RBD) of the spikeprotein of several MERS-CoV clinical isolates. National Center forBiotechnology Information (NCBI)-deposited sequences of MERS-CoVclinical isolates were aligned at amino acids 367-606 and compared tothat of the EMC/2012 strain. Clinical isolates harboring the A431P,S457G, S460F, A482V, L506F, D509G, and V534A substitutions (where theamino acid (single letter designation) preceding the number is that ofthe EMC/2012 strain, and the amino acid (single letter designation)following the number is that of the clinical isolate) are shown in thetable.

TABLE I No variation from EMC/2012 sequence A431P S457G S460F A482VL506F D509G V534A Al-Hasa_1 Riyadh_9 KFU-HKU 1 Qatar3 Riyadh_9 England 1Bisha_1 Riyadh_2 Al-Hasa_2 KFU-HKU 13 Qatar4 England- Riyadh_1 Al-Hasa_3Qatar/2012 Al-Hasa_4 Al-Hasa_12 Al-Hasa_15 Al-Hasa_16 Al-Hasa_17Al-Hasa_18 Al-Hasa_19 Al-Hasa_21 Al-Hasa_25 Buraidah_1 EMC/2012 FRA/UAEHafr-Al-Batin_1 Hafr-Al-Batin_2 Hafr-Al-Batin_6 Jeddah_1 Jordan-N3/2012Munich Riyadh_3 Riyadh_4 Riyadh_5 Riyadh_14 Taif_1 Wadi-Ad-Dawasir_1

In some embodiments, an antagonist is administered before (e.g., atleast 1, 2, 4, 6, 12, 24, 48 hours, 2, 3, 4, 5, 6, or 7 days, or morebefore) a MERS-CoV infection in the rodent. In other embodiments, theantagonist is administered after (e.g., at least 1, 2, 4, 6, 12, 24, 48hours, 2, 3, 4, 5, 6, or 7 days, or more after) a MERS-CoV infection inthe rodent.

In some embodiments, where an antagonist is administered to a rodentafter MERS-CoV infection, a lower viral titer or RNA level (e.g., viralUpE or leader sequence RNA level) in the rodent after administration ofthe antagonist, e.g., lower by at least 5-fold (e.g., at least 5, 10,50, 100, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷-fold or more) compared to acontrol level indicates that the antagonist is effective in treating aMERS-CoV infection. For example, a control level is the viral titer orRNA level in the rodent prior to administration of the antagonist. Inother examples, a control level is the viral titer or RNA level in avirus-infected rodent that is untreated with the antagonist.

In some embodiments, where an antagonist is administered to a rodentprior to MERS-CoV infection, a lower viral titer or RNA level (e.g.,viral UpE or leader sequence RNA level) in the rodent afteradministration of the antagonist, e.g., lower by at least 5-fold (e.g.,at least 5, 10, 50, 100, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷-fold or more)compared to a control level indicates that the antagonist is effectivein preventing a MERS-CoV infection. For example, a control level is theviral titer or RNA level of a rodent infected with MERS-CoV that was nottreated with the antagonist.

In some embodiments, viral RNA levels in a rodent lung can be determinedby extracting RNA from the rodent lung by homogenization in a solutioncontaining phenol, e.g., a solution containing phenol and guanidiniumisothiocyanate (e.g., Trizol® (Life Technologies, Inc)). For example,the lung can be homogenized using a Magnalyzer (Roche) according to themanufacturers' instructions. In some embodiments, levels of MERS-CoV RNAcan be assessed using quantitative PCR (qPCR) using primers that targetthe MERS-CoV genome and/or primers that target the MERS-CoV mRNAtranscript. For example, the Taqman® Fast virus one-step master mix(Applied Biosystems) can be used in qPCR according to the manufacturers'instructions using a duplex of primers obtained from Life Technologiestargeting a region of the genome upstream of the envelope gene (UpE) orthe leader sequence of the nucleocapsid messenger RNA (leader primer),and compared to an endogenous control, such as rodent (e.g., mouse) 18SrRNA. For example, qPCR reactions in Microamp® fast optical reactionplates (Applied Biosystems) can be read on a 7500 fast DX real-time PCRinstrument (Applied Biosystems). In some examples, qPCR data can beanalyzed using the delta Ct method, with an uninfected control set to 1.For example, percent MERS-CoV RNA detected can be expressed relative tolevels of RNA detected in infected mice treated with control antagonist,e.g., isotype-matched control antibodies, in cases where the antagonistis an antibody against MERS-CoV.

In some embodiments, the viral titer in a rodent lung can be determinedby homogenizing the rodent lung in a buffer (e.g., phosphate bufferedsaline (PBS)), centrifuged (e.g., at 10,000 rpm). The supernatant can beanalyzed by plaque assay on mammalian cells, such as Vero cells (e.g.,Vero E6 cells) to quantitate levels of virus remaining after treatmentwith an antagonist. A standard plaque assay can be used, e.g., a plaqueassay described in Page et al. (2012) Induction of alternativelyactivated macrophages enhances pathogenesis during severe acuterespiratory syndrome coronavirus infection, J Virol 86:13334-13349. Insome examples, the Page et al. plaque assay can be modified by leavingplates for 3 days for plaques to appear.

In some embodiments, less inflammation (e.g., interstitial orperi-vascular inflammation), fewer inflammatory cells, and/or lessbronchiolar cuffing, e.g., determined by histological analysis, in alung sample of a rodent treated with an antagonist prior to or afterMERS-CoV infection compared to a control lung sample can indicate thatthe antagonist is effective in treating or preventing a MERS-CoVinfection. For example, a control lung sample can be from a rodentinfected with MERS-CoV that is untreated with an antagonist, e.g.,untreated before and/or after infection.

The rodents described herein are useful for the efficient testing ofdrugs and vaccines for MERS-CoV, e.g., to demonstrate safety andefficacy prior to clinical testing in humans. They permit rapididentification and/or validation of therapeutics/prophylactics, e.g.,within several weeks of testing.

Definitions

Homologous amino acid residues are residues that share similarcharacteristics or properties. Characteristics or properties of an aminoacid residue are based on, e.g., the structure of the polypeptidebackbone, for example, a sheet or helical conformation, the charge orhydrophobicity of the residue, and/or the bulk of the side chain(s). Forexample, homologous residues are similar in side chain properties, e.g.,polarity, charge, size, aromaticity, and/or hydrophobicity.

The term, “about” refers to a stated value plus or minus another amount;thereby establishing a range of values. In certain embodiments, “about”indicates a range relative to a base (or core or reference) value oramount plus or minus up to 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or 0.1%. For example, aboutrefers to a range of +/−5% below and above the recited numbers, e.g.,numbers of nucleotide bases.

The term “operably linked” as used herein refers to positions ofcomponents so described, e.g., nucleotide sequences, are in arelationship permitting them to function in their intended manner.

As used herein, the term “protein” includes polypeptides, peptides,fragments of polypeptides, and fusion polypeptides.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides covalently joined togetherin either single or double-stranded form.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence. In one instance, anendogenous non-human gene or fragment thereof is replaced with acorresponding human gene or fragment thereof. A corresponding human geneor fragment thereof is a human gene or fragment that is an ortholog of,a homolog of, or is substantially identical or the same in structureand/or function, as the endogenous non-human gene or fragment thereofthat is replaced.

As used herein, the term “rodent” refers to any member of the orderRodentia. Examples of rodents include, without limitation, mice, rats,squirrels, prairie dogs, porcupines, beavers, guinea pigs, and hamsters.In one embodiment, a rodent is a rat. In another embodiment, a rodent isa mouse.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The following examples are provided for illustrative purposes only andnot intended to limit the invention in any manner.

EXAMPLES Example 1 Replacement of the Endogenous Mouse Dpp4 Gene with aHuman DPP4 Gene

The 81.8 kb human DPP4 gene containing exon 2 through exon 26 and aportion of the 3′ untranslated region of the human DPP4 gene replaced78.8 kb of the murine Dpp4 gene locus spanning exon 2 through the stopcodon in exon 26. See FIGS. 1A and 1B.

A targeting construct for replacing the mouse with the human DPP4 genein a single targeting step was constructed using VelociGene® geneticengineering technology (see Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human DPP4 DNA wereobtained from bacterial artificial chromosome (BAC) clones RP23-362N15and RP11-68L22, respectively. Briefly, an SgrDI linearized targetingconstruct generated by gap repair cloning containing mouse Dpp4 upstreamand downstream homology arms flanking 82 kb of human DPP4 sequenceextending from intron 1 upstream of exon 2 through exon 26, includingthe stop codon and a portion of the 3′ untranslated region (genomiccoordinates of the entire human DPP4 gene: GRCh38:chr2:162,848,755-162,931,052 (− strand)), and a floxed neo selectioncassette, was electroporated into VGB6 mouse embryonic stem (ES) cells(derived from C57BL/6N mice). Correctly targeted ES cells (MAID 7326)were further electroporated with a transient Cre-expressing vector toremove the drug selection cassette. Targeted ES cell clones without drugcassette (MAID 7327) were introduced into an 8-cell stage SW mouseembryo by the VelociMouse® method (see, U.S. Pat. Nos. 7,294,754,7,576,259, 7,659,442, and Poueymirou et al. (2007) F0 generation micethat are essentially fully derived from the donor gene-targeted ES cellsallowing immediate phenotypic analyses Nature Biotech. 25(1):91-99).VelociMice® (F0 mice fully derived from the donor ES cell) bearing thehumanized DPP4 gene were identified by genotyping for loss of mouseallele and gain of human allele using a modification of allele assay(see, Valenzuela et al. (2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified Dpp4 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse Dpp4 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, TCGCCACTGT GCCTAACATA G (SEQ ID NO:1);upstream reverse primer, CCGGGACTAA ACTGGAACAT TC (SEQ ID NO:2);upstream probe, FAM-TCAGTCAACT TCTTCTGGGT TGTTTCC-BHQ (SEQ ID NO:3);downstream forward primer, CAGCTCTGGT GGAGAACTAG AC (SEQ ID NO:4);downstream reverse primer, GGAGGTCCTC GGTCTTTAGA AG (SEQ ID NO:5);downstream probe, FAM-TCACACTTAG GCTTATAAAC CATTCCCGT-BHQ (SEQ ID NO:6);in which FAM refers to the 5-carboxyfluorescein fluorescent probe andBHQ refers to the fluorescence quencher of the black hole quencher type(Biosearch Technologies). DNA purified from ES cell clones that havetaken up the targeting vector and incorporated in their genomes wascombined with TaqMan™ Gene Expression Master Mix (Life Technologies)according to the manufacturer's suggestions in a 384-well PCR plate(MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies) andcycled in an Applied Biosystems Prism 7900HT, which collectsfluorescence data during the course of the PCRs and determines athreshold cycle (Ct), the fractional PCR cycle at which the accumulatedfluorescence reaches a pre-set threshold. The upstream and downstreamDPP4-specific qPCRs and two qPCRs for non-targeted reference genes wererun for each DNA sample. The differences in the Ct values (Mt) betweeneach DPP4-specific qPCR and each reference gene qPCR were calculated,and then the difference between each

Ct and the median ΔCt for all samples assayed was calculated to obtainΔΔCt values for each sample. The copy number of the DPP4 gene in eachsample was calculated from the following formula: copynumber=2×2^(−ΔΔCt). A correctly targeted clone, having lost one of itsnative copies, will have a Dpp4 gene copy number equal to one.Confirmation that the human DPP4 gene sequence replaced the deletedmouse Dpp4 gene sequence in the humanized allele was confirmed by aTaqMan™ qPCR assay that comprises the following primer-probe sets(written 5′ to 3′): human upstream forward primer, GCGGTCTCCC TCTTCTAACG(SEQ ID NO:7); human upstream reverse primer, GCAAGCCGAG CAGATCAAG (SEQID NO:8); human upstream probe, FAM-ACTCCCACCT GCAAATCCTG CTGC-BHQ (SEQID NO:9); human downstream forward primer, AACCGCACTG GCATATGGA (SEQ IDNO:10); human downstream reverse primer, TACAAGGTAG TCTGGGATTA CTAACAAAA(SEQ ID NO:11); human downstream probe, FAM-ACATTTATCT AGAAAGGCTC-BHQ(SEQ ID NO:12).

The same LONA assay is used to assay DNA purified from tail biopsies formice derived from the targeted ES cells to determine their DPP4genotypes and confirm that the humanized DPP4 allele is transmittedthrough the germline. Two pups heterozygous for the replacement are bredto generate a mouse that is homozygous for the replacement of theendogenous mouse Dpp4 gene by the human DPP4 gene. Pups that arehomozygous for the replacement are used for phenotyping.

The upstream junction of the murine Dpp4 locus and the sequencecontaining the human DPP4 gene is designed to be within 5′-AGGAGAGAAGCCAACAAGAT CATAAGATCA TGCTCAGGGC CAAAATTCAA GGGCTTCTGC (CGTCGACG)GCCTTAGAGA ACTCCAACTG GCGCACTCCA GACGCCACCC CCACCCCCAG CCCGCGGTCTCCCTCTTCTA ACGCACTCCC ACCTGCAAAT (SEQ ID NO:13), wherein the human DPP4sequences are italicized, and the SgrDI restriction site is bracketed.The downstream junction of the sequence containing the human DPP4 geneand the floxed neo selection cassette is designed to be within5′-TTATTCCAGG GAACTATGAT GAGGCTTATA TAAGAACGAA TAAGATCAGA AATATCATTCTGGCAGTTCT TATGGCTCAG ctcgag(ataa cttcgtataa tgtatgctat acgaagttat)atgcatggcc tccgcgccgg gttttggcgc ctcccgcggg (SEQ ID NO:14), wherein thehuman DPP4 sequences are italicized, the neo cassette sequences are inlower case, and the loxP site is bracketed. The downstream junction ofthe sequence of the floxed neo selection cassette and the murine Dpp4locus is designed to be within 5′-atgtctgga(a taacttcgta taatgtatgctatacgaagt tat)gctagta actataacgg tcctaaggta gcgagctagc CAGCATAGCTCTCCATAGCT TATTTAAGAC CACATTTGTT CTCATTATCT CAAAAGTGCA CTGTTAAGATGAAGATCTTA (SEQ ID NO:15), wherein the neo cassette sequences are inlower case, and the loxP site is bracketed. After removal of the neoselection cassette, the junction of the sequence containing the humanDPP4 gene, the loxP site remaining after removal of the neo selectioncassette, and the murine Dpp4 locus is designed to be within5′-TATTCCAGGG AACTATGATG AGGCTTATAT AAGAACGAAT AAGATCAGAA ATATCATTCTGGCAGTTCTT ATGGCTCAG ctcgag(ataa cttcgtataa tgtatgctat acgaagttat)gctagtaact ataacggtcc taaggtagcg agctagcCA GCATAGCTCT CCATAGCTTATTTAAGACCA CATTTGTTCT CATTATCTCA AAAGTGCACT GTTAAGATGA AGATCTTAATAATGTTGCAT TGAGACATTT CAGGCTGCTT TCTCCAGTTT TACACCTGCA ATCCTAACTAAGGATGCCTG TCCCCAGAAC (SEQ ID NO:16), wherein the human DPP4 sequencesare italicized, the neo cassette sequences are in lower case, and theloxP site is bracketed.

FIG. 2 shows the amino acid sequence of DPP4 encoded by the humanizedDPP4 nucleic sequence in MAID 7326/7327 (SEQ ID NO:17) is the same ashuman DPP4 because mouse Dpp4 codon 1, encodes only the first two aminoacids of DPP4, Met and Lys, which are the same as those encoded by humanDPP4 codon 1.

Example 2 Infection of Humanized DPP4 Mice by MERS-CoV

Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) is a newlyemergent virus that causes severe acute respiratory disease. Thereceptor for MERS-CoV is dipeptidyl peptidase IV (DPP4) (see Raj et al.(2013) Dipeptidyl Peptidase 4 is a Functional Receptor for the EmergingHuman Coronavirus-EMC, Nature 495(7440):251-254). In vivo testing ofanti-viral molecules requires an animal model, e.g., a small animalmodel, such as a rodent (e.g., mouse or rat), that is susceptible toMERS-CoV infection. However, recent studies have shown that mouse Dpp4cannot support MERS-CoV infection (see, e.g., Cockrell et al. (2014)Mouse Dipeptidyl Peptidase is Not a Functional Receptor for Middle EastRespiratory Syndrome Coronavirus (MERS-CoV) Infection, J. Virol.88(9):5195-5199; and Coleman et al. (2014) Wild-Type and InnateImmune-Deficient Mice are Not Susceptible to the Middle East RespiratorySyndrome Coronavirus, J. Gen. Virol. 95(2):408-412), at least in partbecause the MERS-CoV Spike protein interacts with human, but not mouse,DPP4 (see, e.g., Coleman et al. (2013); and Raj et al. (2013)). Sequencecomparison of the sequences of mouse and human DPP4 revealed that theamino acids that have previously been identified as contact sitesbetween MERS-CoV spike (S) protein and its receptor differ between thetwo species. In addition, expression of human DPP4 in mouse cells allowsfor MERS-CoV virus entry and propagation, indicating that entry of thevirus is the limiting step in infection of mouse cells, and that thelack of interaction between mouse DPP4 and the MERS-CoV glycoproteindefines the species tropism in vitro. See, e.g., Lu et al. (2013)Molecular basis of binding between novel human coronavirus MERS-CoV andits receptor CD26, Nature. 500(7461):227-31; and Cockrell et al. (2014)Mouse Dipeptidyl Peptidase is Not a Functional Receptor for Middle EastRespiratory Syndrome Coronavirus (MERS-CoV) Infection, J. Virol.88(9):5195-5199.

As a consequence, normal mouse strains cannot be used to measure theefficacy of therapeutics targeting MERS-CoV. Zhao et al. (2014) RapidGeneration of a Mouse Model for Middle East Respiratory Syndrome, Proc.Natl. Acad. Sci. USA 111(13):4970-4975 have expressed human DPP4 in miceby adenovirus transduction, thereby allowing for MERS-CoV infection.However, this adenovirus model has several limitations, including: (a)the virus is cleared rapidly from infected mice; (b) there is a loss ofhuman DPP4 expression over time; (c) the tissue distribution ofvirally-transduced DPP4 does not reflect expression seen in mice orhumans; and (d) adenovirus infection induces an interferon response.

To generate a mouse model for MERS-CoV infection, humanized DPP4 micewere generated as described above in Example 1. As shown in FIG. 1B,exons 2 through 26 of mouse Dpp4 were replaced by the correspondingsequences of human DPP4. Because the remaining mouse Dpp4 coding exon 1encodes only the first two amino acids of Dpp4, Met and Lys, which arethe same as those in corresponding human DPP4 exon 1, the DPP4 proteinexpressed in humanized DPP4 mice is completely human (see FIG. 2, SEQ IDNO:17). Thus, humanized DPP4 mice express a fully human DPP4 under thecontrol of the endogenous mouse regulatory sequences, i.e., 5′ flankingregion (promoter and enhancer(s)), and 3′ untranslated region sequences.It is expected that the humanized DPP4 is expressed in the same cellsand tissues, and at the same or similar levels, as mouse Dpp4 isexpressed in wild-type mice lacking human DPP4 nucleic acid sequences.

Two F0 humanized DPP4 mice were infected intranasally with MERS-CoV(Jordan strain) or mock treated with PBS. Four days post-infection,MERS-CoV RNA was quantified in the lungs by real-time PCR using primersspecific for the replicative form of the MERS-CoV genome. Data wasnormalized to the amount of PCR product obtained from the lungs of themock-infected mice (arbitrarily set at 1). FIG. 3 shows that MERS-CoVRNA could be amplified from lungs of the MERS-CoV-infected humanizedDPP4 mice. H&E staining was also performed using lung tissue from mock-and MERS-CoV-infected mice.

FIG. 4 shows that MERS-CoV infection of humanized DPP4 mice did notaffect the airway, but resulted in thickening of the walls of thealveoli and less space between alveolar cells, indicating inflammationin the lungs associated with MERS-CoV infection.

In addition, 6 to 8 week old mice were inoculated intranasally withMERS-CoV, e.g., 2×10⁵ pfu of MERS-CoV (Jordan). No mortality or clinicalsigns of disease were observed up to day 4 after inoculation. On days 2and 4 post-inoculation, mice were euthanized and their lungs weredissected. To obtain virus RNA levels, lungs were homogenized inTrizol®, RNA extracted, and analyzed by real-time PCR using primersspecific to MERS-CoV (FIGS. 10A and 10B). A set of primers was specificto a region of the viral genome upstream of the envelope gene (UpE), andanother set of primers was specific to the leader sequence of thenucleocapsid mRNA (leader primer). Mouse 18S rRNA was used as endogenouscontrol.

To obtain virus titers, lungs were homogenized in phosphate bufferedsaline (PBS), clarified by centrifugation, and titered on Vero E6 cells(FIG. 10C). For example, the supernatant was analyzed by a plaque assayon VeroE6 cells to quantitate the levels of virus present in the lungs.For example, plaque assays were performed as described in Page et al.(2012) Induction of alternatively activated macrophages enhancespathogenesis during severe acute respiratory syndrome coronavirusinfection, J Virol 86:13334-13349, with plates left for 3 days forplaques to appear.

Robust MERS-CoV replication in the lungs was evident at 2 and 4 dayspost-infection. RNA quantification, using a primer set specific forMERS-CoV leader, which was designed to only amplify replicatingMERS-CoV, demonstrated high levels of MERS-CoV replicating RNA in lungscollected at day 2, and these levels were maintained through day 4post-infection (FIGS. 10A-B). Plaque assay of lung homogenate on Vero E6cells quantified MERS-CoV (Jordan) levels of ˜7.27×10⁴ pfu/mL lung atday 2 and ˜3.75×10⁵ pfu/mL lung at 4 days post-infection (FIG. 10C),demonstrating active replication of MERS-CoV in the lungs of theinfected humanized DPP4 mice.

Also, lungs from humanized DPP4 mice intranasally inoculated with eitherMERS-CoV (Jordan strain) or PBS (mock infected) were analyzed forpathological changes (FIG. 10D). At day 2 post-infection,peri-bronchiolar inflammation was evident with alterations inbronchiolar cell structure found throughout the lungs. Minimalperi-vascular inflammation or effects on alveolar structures wereobserved at this time point. At 4 days post-infection, interstitialinfiltration was observed with peri-vascular cuffing and extensivealveolar thickening. Bronchiolar alterations were present as well. SeeFIG. 10D. This pathology is consistent with the radiographic findings ofdevelopment of interstitial pneumonia and significant lung disease seenin humans with MERS-CoV.

The above data shows that humanized DPP4 mice, such as those describedherein, are susceptible to MERS-CoV infection. The data also demonstratethat the humanized DPP4 mice described herein are an in vivo model ofMERS-CoV infection that recapitulates the pathology, e.g., pathologicalsequelae, that is seen in MERS-CoV infection of humans.

Thus, humanized DPP4 mice are a robust model of MERS-CoV that is usefulto assess MERS-CoV treatment in vivo. For example, the humanized DPP4mice are appropriate host animals to measure the pharmacokinetics,pharmacodynamics and therapeutic efficacy of therapeutic molecules thattarget MERS-CoV.

FIG. 5 shows a protein sequence alignment of mouse Dpp4 (SEQ ID NO: 25)and human DPP4 (encoded by the 7326/7327 transgenic mice) (SEQ ID NO:26).

Next, a dose-response study of weight as a function of timepost-infection of MERS-CoV was conducted in humanized DPP4 mice. Micewere infected with either MERS-CoV (Jordan strain) or PBS (mockinfected) as described above and were analyzed for weight loss, which isa sign of productive infection, over a period of seven days. As shown inFIG. 14, humanized DPP4 mice exhibited productive infection (i.e.,manifested disease pathology), with weight loss beginning 4 dayspost-infection. Four to five mice were used per group. FIG. 15 showsthat mice that were heterozygotic for the humanized DPP4 allele wereequally susceptible to infection by MERS-CoV, as they exhibited asimilar degree of weight loss when compared to homozygotes. This findingis significant because it indicates that studies can be conducted usingheterozygous mice. Additionally, use of heterozygous mice avoids anypotential issue related to functional mouse Dpp4 knockouts that couldpotentially be present in homozygous humanized DPP4 mice.

The lungs of these mice were also examined histologically forinflammation according to the methods described above. As shown in FIG.16, hDPP4 mice exposed to a high dose of virus (1×10exp6 pfu/mouse)exhibited increased pathology relative to PBS controls.

Example 3 Generation of Transgenic Mice Containing the Human DPP4 GeneUsing Random Insertion of BACs

Transgenic mice were generated that contain the human DPP4 gene, forwhich the sequence and genomic information is shown in FIG. 6. Twodifferent overlapping BACs containing the human DPP4 gene were used: BACRP11-68L22 and BAC RP11-345J9 (FIG. 7). Both BACs contained the codingregion of the human DPP4 gene, as well as over 40 kb of promoter regionupstream of the ATG start codon of the DPP4 gene and several kilo basesdownstream of the stop codon of the DPP4 gene (FIG. 7).

To generate the BAC transgenic mice, each BAC DNA was electroporatedinto VGB6 mouse embryonic stem (ES) cells (derived from C57BL/6N mice).ES cells containing the coding region of the human DPP4 gene, as well aspromoter regions of the gene, were introduced into an 8-cell stage SWmouse embryo by the SW mouse embryo by the VelociMouse® method (see,e.g., U.S. Pat. Nos. 7,294,754, 7,576,259, 7,659,442, and Poueymirou etal. (2007) F0 generation mice that are essentially fully derived fromthe donor gene-targeted ES cells allowing immediate phenotypic analysesNature Biotech. 25(1):91-99).

Human gain of allele assays were used to screen ES cell clones for onesthat contained copies of the human DPP4 gene along with promoter regionsof the gene. Human gain of allele assays were also used to identifyVelociMice® (F0 mice fully derived from the donor ES cell) bearing thehumanized DPP4 gene along with promoter regions of the gene.

Briefly, genomic DNA was extracted from ES cell clones using standardmethods and tested in a Taq Man™ quantitative PCR (q PCR) assay usingtwo sets of primer-probes to detect a human DNA sequence upstream (7333hTU) and downstream hTD) of the human DPP4 coding sequence (FIG. 8). Thelocations within the human DPP4 gene and flanking regions (e.g.,promoter regions) at which each primer-probe set annealed is shown inFIG. 7. A fluorescent read-out above background in the TaqMan™ qPCRassay indicated the presence of the human DPP4 gene and at least 40 kbof the 5′ flanking region of the human DPP4 gene that had beenintegrated into the transgenic mouse genome.

The 7333 hTU primer-probe set (written 5′ to 3′) was: human upstreamforward primer, TGGCTTATTCTCTATTCCTCACCTA (SEQ ID NO: 18); humanupstream probe, FAM-TGCTTTCCCTCCTCCCTTCTGA-BHQ (SEQ ID NO: 19); humanupstream reverse primer, GGCCTTAGCCCAGAAACTG (SEQ ID NO: 20). The 7333hTD primer-probe set (written 5′ to 3′) was: human downstream forwardprimer, TGCAGACTTGTCTTGACATTCATA (SEQ ID NO: 21); human downstreamprobe, CAL-AGCCTCTGCAGACACAGGAATGGC-BHQ (SEQ ID NO: 22); and humandownstream reverse primer, TCTGGGCACTGGTGTACTC (SEQ ID NO: 23); in whichFAM and CAL refer to the 5-carboxyfluorescein and CAL Orange fluorescentprobes, respectively, and BHQ refers to the fluorescence quencher of theblack hole quencher type (Biosearch Technologies).

For example, genomic DNA from ES cell clones was combined with TaqMan™Gene Expression Master Mix (Life Technologies) according to themanufacturer's suggestions in a 384-well PCR plate (MicroAmp™ Optical384-Well Reaction Plate, Life Technologies) and cycled in an AppliedBiosystems Prism 7900HT, which collects fluorescence data during thecourse of the PC Rs and determines a threshold cycle (Ct), thefractional PCR cycle at which the accumulated fluorescence reaches apre-set threshold. The upstream and downstream DPP4-specific qPCRs andtwo qPCRs for non-DPP4 reference genes were run for each DNA sample. Thedifferences in the Ct values (

Ct) between each DPP4-specific qPCR and each reference gene qPCR werecalculated, and then the difference between each at and the median

Ct for all samples assayed was calculated to obtain

Ct values for each sample. The copy number of the DPP4 gene in eachsample was calculated from the following formula: copy number=2×2⁻

^(Ct). A clone containing at least one copy of the human DPP4 pluspromoter regions integrated into the chromosome had a DPP4 gene copynumber equal to or greater than one.

The same human gain of allele assay was used to assay DNA purified fromtail biopsies for mice derived from the ES cells to confirm that thehumanized DPP4 allele along with the human 5′ flanking regions weretransmitted through the germline.

Using the BAC insertion and screening methods described herein, twotransgenic mice with DNA encoding human DPP4 were confirmed. BACRP11-68L22 was used to generate ES cell clones and transgenic micereferred to as MAID 7333, and BAC RP11-345J9 was used to generate EScell clones and transgenic mice referred to as MAID 7334.

The protein encoded by the humanized DPP4 nucleic acid sequence in theMAID 7333 and 7334 mice had the amino acid sequence shown in FIG. 9 (SEQID NO: 24), which is the same as human DPP4 (as encoded by thetranscript, NM_001935.3).

Example 4 Treatment of Humanized DPP4 Mice that were Infected with theMERS-CoV Virus

Transgenic mice with the humanized DPP4 gene and flanking promoterregions were tested for their ability to be infected by MERS-CoV and toserve as a model for assessing therapeutic molecules for treating orpreventing MERS-CoV.

Transgenic MAID 7333 mice (e.g., generated by the methods described inExample 3) were treated with 200 μg of antibodies directed againstMERS-CoV spike protein or isotype controls by intraperitoneal injection(ip). One day after antibody injection, the mice were infectedintranasally with MERS-CoV. Four days after infection, lungs of the micewere harvested, and viral RNA levels were measured using real-time PCR(RT-PCR). In particular, levels of the genomic RNA (UpE) or replicatingRNA (leader) (specific for the replicative form of the MERS-CoV genome)of MERS-CoV were measured.

The RT-PCR data is shown in the table below.

Antibody UpE¹ Leader¹ Anti-MERS-CoV spike protein 1 (Ab 1) 0.3568395620.273565089 Anti-MERS-CoV spike protein 2 (Ab 2) 0.254493202 0.206006238Anti-MERS-CoV spike protein 3 (Ab 3) 1.989548316 1.112094283 (IgG1)isotype control 104.0889287 101.2578723 (IgG4) isotype control 100 100¹Averages (% of isotype control)

Treatment of transgenic mice with the antibodies decreased viral RNAlevels (both UpE and Leader) by about 50-fold to about 500-fold.

The data described herein show that transgenic mice generated bytargeted integration methods (Example 1) and random BAC insertionmethods (Example 3) with human DPP4 were susceptible to infection byMERS-CoV. In addition, anti-MERS-CoV antibodies blocked infection intransgenic mice in vivo. Thus, transgenic mice with human DPP4 (e.g.,generated by the methods described herein) are useful for evaluating theefficacy of therapeutics (e.g., antibodies) that target the MERS-CoVvirus.

Example 5 Prophylactic Effects of Anti-MERS-CoV Antibodies on MERS-CoVInfection in Humanized DPP4 Mice

The humanized DPP4 mice described herein were used to evaluate theprophylactic capability of the two monoclonal antibodies in vivo. Micewere i.p. injected with a dose range of anti-MERS-CoV antibodies 200 μg,20 μg or 2 μg of anti-MERS-CoV spike protein antibody 2 (Ab 2),anti-MERS-CoV spike protein antibody 4 (Ab 4), or 200 μg of human IgG 1(hIgG 1) isotype control antibody—at 24 hours before intranasalinfection with 1×10⁵ pfu of MERS-CoV (Jordan strain). Ab 2 and Ab 4 werefully human anti-MERS-CoV spike protein antibodies. RNA was extractedfrom the mouse lungs and analyzed by quantitative PCR as describedabove. For example, qPCR data was analyzed using the delta Ct method,with an uninfected control set to 1. Percent MERS-CoV RNA detected wasexpressed relative to levels of RNA detected in infected mice treatedwith isotype-matched control antibodies (FIGS. 11A-B). Also, viraltiters from mouse lungs were determined as described above.

Both antibodies significantly decreased MERS-CoV specific RNA levels inthe lungs by over 2 logs at the 200 μg per mouse dose, compared to theisotype-matched control antibody (FIGS. 11A-B). Ab 2 was more effectiveat reducing MERS-CoV RNA levels at the 20 μg dose compared to Ab 4 atthe same dose. The 2 μg dosing of either antibody was ineffective atreducing viral RNA levels compared to isotype control treated mice. WhenMERS-CoV titer was analyzed in the lungs (FIG. 11C), both the 200 μg and20 μg dose of Ab 2 reduced virus levels to near the level of detectionin the assay (2×10³ pfu/ml). Ab 4 was equally efficient at the 200 μgdose as Ab 2, while the 20 μg and 2 μg doses displayed a dose dependentinhibition of viral inhibition. These data show that anti-MERS-CoVantibodies, e.g., Ab 2 and Ab 4, effectively blocked MERS-CoV infectionin vivo.

Histological analysis was also performed at 4 days post-infection onlungs from mice treated at 24 hours pre-infection with Ab 2, Ab 4, orhIgG 1 isotype control antibody (FIG. 12A). For example, the degrees ofinterstitial, peribronchiolar, and perivascular inflammation were scoredfrom 0 to 5. Other histologic features, such as the presence ofbronchiolar epithelial and alveolar damage, pleural changes and theextent of peribronchovascular inflammation, were also analyzed. Anoverall inflammatory score for each mouse was averaged for eachexperimental group, and the scores were presented as average scores ofall mice in each group and time point (FIG. 12B).

Lungs from mice pre-treated with hIgG 1 isotype control mice displayedsignificant lung pathology with increased interstitial inflammation,perivascular cuffing, and thickening of alveolar septa. Mice treatedwith 200 μg of either Ab 2 or Ab 4 had reduced inflammation with minimalfoci of inflammatory cells in the interstitium, minor bronchiolarcuffing, and less alveolar wall thickening. In mice pre-treated with 20μg of Ab 2 and Ab 4, there were moderate levels of perivascular cuffingand interstitial inflammation compared to the higher dose antibodygroup. The 2 μg antibody pre-treated group had similar pathology to thehIgG1 isotype control, displaying significant interstitial inflammationand predominant peri-vascular inflammation. Blinded histological scoringdemonstrated reduced inflammation scores for treated mice (FIG. 12B).These findings demonstrate that anti-MERS-CoV antibodies, such as Ab 2and Ab 4, confer a dose-dependent reduction in lung pathology followingMERS-CoV infection, corroborating viral RNA levels and virus titers inthe mice.

Thus, anti-MERS-CoV antibodies, such as Ab 2 and Ab 4, were effective inan in vivo model of MERS-CoV infection—the antibodies blocked MERS-CoVinfection and disease in vivo when injected before infection, e.g., 1day before infection.

Example 6 Antibody Treatment of Humanized DPP4 Mice that have beenInfected with MERS-CoV

To determine the therapeutic effect (e.g., ability to inhibit MERS-CoVreplication and lung pathology after infection) of anti-MERS-CoVantibodies (e.g., Ab 2 or Ab 4), humanized DPP4 mice were infected withMERS-CoV. At 24 hours post-infection, the mice were injected i.p. witheither 500 μg of hIgG1 isotype control or Ab 2 at 500 μg or 200 μg. At 4days post-infection, mice were euthanized and mouse lungs analyzed forviral RNA, virus titer, and lung pathology. Both the 500 μg and 200 μgdoses of Ab 2 reduced viral RNA levels by about 10 fold in the lungs ofmice compared to control antibody treated mice (FIGS. 13A-B). Lungtiters of the same mice demonstrated significant reduction in virallevels in the lungs, with a greater than 2 log reduction at day 4post-infection (FIG. 13C). These data demonstrate that after infection,e.g., 24 hours post-infection, an anti-MERS-CoV antibody (e.g., Ab 2)significantly inhibited viral replication.

Histological analysis was also performed on mice treated 24 hourspost-infection with hIgG1 control antibody, 500 μg Ab 2, or 200 μg Ab 2(FIGS. 14A-B). Mice treated with control antibody displayed similarpathology to the controls in Examples 2 and 5, with significantinterstitial inflammation, peri-vascular cuffing, and thickening ofalveolar septa. Mice treated with either 200 μg or 500 μg of Ab 2 hadminimal interstitial inflammation with reduced and only focalperi-vascular inflammation throughout the lungs. Blinded histologicalscoring demonstrated reduced inflammation scores for treated mice (FIG.14B). The data demonstrate that therapeutic doses of anti-MERS-CoVantibodies (e.g., Ab 2) reduced MERS-CoV induced lung pathology evenwhen given after infection, e.g., 24 hours post-infection.

1. A humanized DPP4 rodent embryo comprising a humanization of anendogenous rodent Dpp4 gene, wherein the humanization comprises areplacement at the endogenous rodent Dpp4 locus of at least one exon ofthe endogenous rodent Dpp4 gene with a nucleic acid sequence encoding atleast one exon of a human DPP4 gene to form a modified DPP4 gene,wherein expression of the modified DPP4 gene is under control of rodentregulatory elements at the endogenous rodent Dpp4 locus, and wherein therodent is a mouse or a rat.
 2. The embryo of claim 1, wherein the humanDPP4 gene encoding a human or humanized DPP4 protein comprises exon 2through exon 26 of the human DPP4 gene.
 3. The embryo of claim 2,wherein the humanized DPP4 protein comprises the extracellular domain ofthe human DPP4 protein.
 4. The embryo of claim 3, wherein the humanizedDPP4 protein comprises the transmembrane domain and cytoplasmic domainof the rodent Dpp4 protein.
 5. The embryo of claim 1, wherein the rodentis a mouse that is incapable of expressing a mouse Dpp4 protein.
 6. Theembryo of claim 1, wherein the humanization of the endogenous rodentDpp4 gene is heterozygous.
 7. The embryo of claim 1, wherein thehumanization of the endogenous rodent Dpp4 gene is homozygous.
 8. Theembryo of claim 1, wherein the rodent is a mouse and wherein the mouseis: (a) a C57BL strain selected from the group consisting of C57BL/A,C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/01a; (b) a 129strain selected from the group consisting of 129P1, 129P2, 129P3, 129X1,129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH,129S6 (129/SvEvTac), 129S7, 129S8, 129T1, and 129T2; (c) a mixture of a129 strain and a C57BL/6 strain; (d) a mix of 129 strains; or (e) a mixof BL/6 strains.
 9. A rodent host embryo comprising a donor cell thatcomprises a humanization of an endogenous rodent Dpp4 gene, wherein thehumanization comprises a replacement at the endogenous rodent Dpp4 locusof at least one exon of the endogenous rodent Dpp4 gene with a nucleicacid sequence encoding at least one exon of a human DPP4 gene to form amodified DPP4 gene, wherein expression of the modified DPP4 gene isunder control of rodent regulatory elements at the endogenous rodentDpp4 locus, and wherein the rodent is a mouse or a rat.
 10. The hostembryo of claim 9, wherein the human DPP4 gene encoding a human orhumanized DPP4 protein comprises exon 2 through exon 26 of the humanDPP4 gene.
 11. The host embryo of claim 10, wherein the humanized DPP4protein comprises the extracellular domain of the human DPP4 protein.12. The host embryo of claim 11, wherein the humanized DPP4 proteincomprises the transmembrane domain and cytoplasmic domain of the rodentDpp4 protein.
 13. The host embryo of claim 9, wherein the rodent is amouse that is incapable of expressing a mouse Dpp4 protein.
 14. The hostembryo of claim 9, wherein the humanization of the endogenous rodentDpp4 gene is heterozygous.
 15. The host embryo of claim 9, wherein thehumanization of the endogenous rodent Dpp4 gene is homozygous.
 16. Arodent egg comprising an ectopic non-human animal chromosome, whereinthe ectopic non-human animal chromosome comprises a humanization of aendogenous rodent Dpp4 gene, wherein the humanization comprises areplacement at the endogenous rodent Dpp4 locus of at least one exon ofthe endogenous rodent Dpp4 gene with a nucleic acid sequence encoding atleast one exon of a human DPP4 gene to form a modified DPP4 gene,wherein expression of the modified DPP4 gene is under control of rodentregulatory elements at the endogenous rodent Dpp4 locus, and wherein therodent is a mouse or a rat.
 17. The rodent egg of claim 16, wherein thehuman DPP4 gene encoding a human or humanized DPP4 protein comprisesexon 2 through exon 26 of the human DPP4 gene.
 18. The rodent egg ofclaim 17, wherein the humanized DPP4 protein comprises the extracellulardomain of the human DPP4 protein.
 19. The rodent egg of claim 18,wherein the humanized DPP4 protein comprises the transmembrane domainand cytoplasmic domain of the rodent Dpp4 protein.
 20. The rodent egg ofclaim 16, wherein the rodent is a mouse that is incapable of expressinga mouse Dpp4 protein.
 21. The rodent egg of claim 16, wherein thehumanization of the endogenous rodent Dpp4 gene is heterozygous.
 22. Therodent egg of claim 16, wherein the humanization of the endogenousrodent Dpp4 gene is homozygous.
 23. A rodent comprising a germ-linemodification, wherein said germ line modification comprises ahumanization of a endogenous rodent Dpp4 gene, wherein the humanizationcomprises a replacement at the endogenous rodent Dpp4 locus of at leastone exon of the endogenous rodent Dpp4 gene with a nucleic acid sequenceencoding at least one exon of a human DPP4 gene to form a modified DPP4gene, wherein expression of the modified DPP4 gene is under control ofrodent regulatory elements at the endogenous rodent Dpp4 locus, whereinthe rodent is a mouse or a rat.
 24. The rodent of claim 23, wherein thehuman DPP4 gene encoding a human or humanized DPP4 protein comprisesexon 2 through exon 26 of the human DPP4 gene.
 25. The rodent of claim24, wherein the humanized DPP4 protein comprises the extracellulardomain of the human DPP4 protein.
 26. The rodent of claim 25, whereinthe humanized DPP4 protein comprises the transmembrane domain andcytoplasmic domain of the rodent Dpp4 protein.
 27. The rodent of claim23, wherein the rodent is a mouse that is incapable of expressing amouse Dpp4 protein.
 28. The rodent of claim 23, wherein the humanizationof the endogenous rodent Dpp4 gene is heterozygous.
 29. The rodent ofclaim 23, wherein the humanization of the endogenous rodent Dpp4 gene ishomozygous.
 30. The rodent of claim 23, wherein the rodent is a mouseand wherein the mouse is: (a) a C57BL strain selected from the groupconsisting of C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6,C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, andC57BL/01a; (b) a 129 strain selected from the group consisting of astrain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, and 129T2; (c) a mixture of a 129 strain and aC57BL/6 strain; (d) a mix of 129 strains; or (e) a mix of BL/6 strains.